Name: Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade
Brand: RabMAb
SKU: ab176916
Rating: 4 (4 reviews)

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Primary

Secondary

This product Rabbit Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)
PepArr, ChIP, ICC/IF, IHC-P, WB, Dot blot

Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
ICC/IF, Flow Cyt, IHC-P, ELISA, IHC-Fr

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Product name

Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade
See all Histone H3 primary antibodies
Description

Rabbit monoclonal [EPR16601] to Histone H3 (tri methyl K9) - ChIP Grade
Host species

Rabbit
Tested applications

Suitable for: PepArr, ChIP, ICC/IF, IHC-P, WB, Dot blotmore details
Species reactivity

Reacts with: Mouse, Rat, Human
Immunogen

Synthetic peptide within Human Histone H3 aa 1-100 (tri methyl K9). The exact sequence is proprietary.
Database link: P68431
Positive control
- WB: HeLa and NIH/3T3 whole cell lysates. IHC-P: Human colon, mouse liver and rat liver tissues. ICC/IF: HeLa and NIH/3T3 cells.
General notes

Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab^® patents.
This product is a recombinant rabbit monoclonal antibody.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
Storage buffer

Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR16601
Isotype

IgG
Research areas

Alternative Versions
- Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - BSA and Azide free (ab232324)
Compatible Secondaries
- Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
- Goat Anti-Rabbit IgG H&L (HRP) (ab97051)
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
Recombinant Protein
- Recombinant human Histone H3 protein (Active) (ab198757)

Our Abpromise guarantee covers the use of ab176916 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application	Abreviews	Notes
PepArr		Use at an assay dependent concentration.
ChIP		Use 2 µg for 25 µg of chromatin.
ICC/IF		1/2000.
IHC-P		1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB		1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
Dot blot		1/1000.

Function

Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.
Sequence similarities

Belongs to the histone H3 family.
Developmental stage

Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
Post-translational
modifications

Acetylation is generally linked to gene activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-19 (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me).
Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription.
Asymmetric dimethylation at Arg-18 (H3R17me2a) by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) by PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the 3' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on active promoters.
Methylation at Lys-5 (H3K4me), Lys-37 (H3K36me) and Lys-80 (H3K79me) are linked to gene activation. Methylation at Lys-5 (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are linked to gene repression. Methylation at Lys-10 (H3K9me) is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 (H3S10ph) and acetylation of H3 and H4. Methylation at Lys-5 (H3K4me) and Lys-80 (H3K79me) require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are enriched in inactive X chromosome chromatin.
Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase because it enables the transcription of genes following external stimulation, like mitogens, stress, growth factors or UV irradiation and result in the activation of genes, such as c-fos and c-jun. Phosphorylation at Ser-11 (H3S10ph), which is linked to gene activation, prevents methylation at Lys-10 (H3K9me) but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 (H3S10ph) is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 (H3S28ph) by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation at Thr-7 (H3T6ph) by PRKCBB is a specific tag for epigenetic transcriptional activation that prevents demethylation of Lys-5 (H3K4me) by LSD1/KDM1A. At centromeres, specifically phosphorylated at Thr-12 (H3T11ph) from prophase to early anaphase, by DAPK3 and PKN1. Phosphorylation at Thr-12 (H3T11ph) by PKN1 is a specific tag for epigenetic transcriptional activation that promotes demethylation of Lys-10 (H3K9me) by KDM4C/JMJD2C. Phosphorylation at Tyr-42 (H3Y41ph) by JAK2 promotes exclusion of CBX5 (HP1 alpha) from chromatin.
Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination (By similarity). Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins.
Cellular localization

Nucleus. Chromosome.
Target information above from: UniProt accession P68431 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 8350 Human
- Entrez Gene: 8351 Human
- Entrez Gene: 8352 Human
- Entrez Gene: 8353 Human
- Entrez Gene: 8354 Human
- Entrez Gene: 8355 Human
- Entrez Gene: 8356 Human
- Entrez Gene: 8357 Human
- Entrez Gene: 8358 Human
- Entrez Gene: 8968 Human
- Entrez Gene: 319152 Mouse
- Entrez Gene: 319153 Mouse
- Entrez Gene: 360198 Mouse
- Entrez Gene: 97908 Mouse
- Entrez Gene: 100364501 Rat
- Entrez Gene: 100365669 Rat
- Entrez Gene: 291159 Rat
- Entrez Gene: 679994 Rat
- Entrez Gene: 680511 Rat
- Entrez Gene: 682330 Rat
- Omim: 602810 Human
- SwissProt: P68431 Human
- SwissProt: P68433 Mouse
- SwissProt: Q6LED0 Rat
- Unigene: 132854 Human
- Unigene: 247813 Human
- Unigene: 247814 Human
- Unigene: 248176 Human
- Unigene: 443021 Human
- Unigene: 484990 Human
- Unigene: 532144 Human
- Unigene: 533292 Human
- Unigene: 546315 Human
- Unigene: 586261 Human
- Unigene: 591778 Human
- Unigene: 221301 Mouse
- Unigene: 261657 Mouse
- Unigene: 377874 Mouse
- Unigene: 390558 Mouse
- Unigene: 397328 Mouse
- Unigene: 138090 Rat
see all
Alternative names
- H3 histone family member E pseudogene antibody
- H3 histone family, member A antibody
- H3/A antibody
- H31_HUMAN antibody
- H3F3 antibody
- H3FA antibody
- Hist1h3a antibody
- HIST1H3B antibody
- HIST1H3C antibody
- HIST1H3D antibody
- HIST1H3E antibody
- HIST1H3F antibody
- HIST1H3G antibody
- HIST1H3H antibody
- HIST1H3I antibody
- HIST1H3J antibody
- HIST3H3 antibody
- histone 1, H3a antibody
- Histone cluster 1, H3a antibody
- Histone H3 3 pseudogene antibody
- Histone H3.1 antibody
- Histone H3/a antibody
- Histone H3/b antibody
- Histone H3/c antibody
- Histone H3/d antibody
- Histone H3/f antibody
- Histone H3/h antibody
- Histone H3/i antibody
- Histone H3/j antibody
- Histone H3/k antibody
- Histone H3/l antibody
see all

ChIP - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 2µg of ab176916 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
Western blot - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

All lanes : Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 15 kDa
Observed band size: 15 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: Lane 1: 1 second; Lane 2: 4 seconds.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H3 (tri methyl K9) with ab176916 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human colon is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Histone H3 (tri methyl K9) with ab176916 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab176916 at 1/2000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.
Dot Blot - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

Dot blot analysis of Histone H3 (tri methyl K9) peptide (Lane 1), Histone H3K9 unmodified peptide (Lane 2), Histone H3 (crotonyl K4) peptide (Lane 3) and Histone H3K4 unmodified peptide (Lane 4) labeled using ab176916 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/1000 dilution.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 3 minutes.
Peptide Array - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

ab176916 was tested in Peptide array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).

Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.

The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling Histone H3 (tri methyl K9) with ab176916 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse liver is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

Immunohistochemical analysis of paraffin-embedded rat liver tissue labeling Histone H3 (tri methyl K9) with ab176916 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on rat liver is observed. Counter stained with Hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Histone H3 (tri methyl K9) with ab176916 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue).
Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab176916 at 1/2000 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunohistochemistry protocols
Immunocytochemistry & immunofluorescence protocols
Western blot protocols

Datasheet

This product has been referenced in:

López-Ozuna VM et al. Prolactin modulates TNBC aggressive phenotype limiting tumorigenesis. Endocr Relat Cancer N/A:N/A (2019). Read more (PubMed: 30640712) »
Li QL et al. The hyper-activation of transcriptional enhancers in breast cancer. Clin Epigenetics 11:48 (2019). Read more (PubMed: 30867030) »

See all 4 Publications for this product

Publishing research using ab176916? Please let us know so that we can cite the reference in this datasheet.

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1-4 of 4 Abreviews or Q&A

Application

Flow Cytometry

Sample

Mouse Cell (T lymphocytes)

Permeabilization

Yes - eBioscience FoxP3/TF staining kit

Gating Strategy

Gated on CD3+/Thy+ cells

Specification

T lymphocytes

Preparation

Cell harvesting/tissue preparation method: Extracted from spleen, by magnetic negative selection
Sample buffer: FACS buffer (PBS + 2% SVF + 0.5 mM EDTA + 0.01% azide)

Fixation

Formaldehyde

Abcam user community

Verified customer

Submitted Jun 20 2017

Application

Western blot

Sample

Mouse Cell lysate - whole cell (mouse embryonic stem cells and HEK293 cells)

Gel Running Conditions

Reduced Denaturing (18)

Loading amount

200000 cells

Treatment

HEK293 cells transfected with SETDB1

Specification

mouse embryonic stem cells and HEK293 cells

Blocking step

(agent) for 12 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Dec 30 2016

Application

Western blot

Sample

Human Cell lysate - nuclear (Melanoma)

Gel Running Conditions

Reduced Denaturing (15%)

Loading amount

3000 cells

Specification

Melanoma

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Dec 23 2016

Application

Western blot

Sample

Human Purified protein (K-562)

Gel Running Conditions

Reduced Denaturing (18% gel)

Loading amount

0.5 µg

Specification

K-562

Blocking step

Odyssey Blocking Buffer (TBS) (LI-COR) 1:1 mixture of Licor blocking buffer and 1X PBS as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 25°C

Dr. Ragnhild Eskeland

Verified customer

Submitted Oct 27 2016

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
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Recombinant Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade (ab176916)

Overview

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Datasheets and documents

References

This product has been referenced in:

Customer reviews and Q&As

Flow Cytometry abreview for Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade

Western blot abreview for Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade

Western blot abreview for Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade

Western blot abreview for Anti-Histone H3 (tri methyl K9) antibody [EPR16601] - ChIP Grade